G-protein coupled receptors (GPCRs) are proteins responsible for transducing a signal within a cell. GPCRs have usually seven transmembrane domains. Upon binding of a ligand to an extra-cellular portion or fragment of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property or behavior of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signalling system that connects the state of intra-cellular second messengers to extra-cellular inputs.
GPCR genes and gene products can modulate various physiological processes and are potential causative agents of disease. The GPCRs seem to be of critical importance to both the central nervous system and peripheral physiological processes.
The GPCR protein superfamily is represented by five families: Family I, receptors typified by rhodopsin and the beta2-adrenergic receptor and currently represented by over 200 unique members; Family II, the parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family, Family IV, the CAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptor such as STE2.
G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors (receptors containing seven transmembrane domains) for signal transduction. Indeed, following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits.
The GTP-bound form of the α, β and γ-subunits typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g. by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
More than 20 different types of α-subunits are known in humans. These subunits associate with a small pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish et al., Molecular Cell Biology (Scientific American Books Inc., New York, N.Y., 1995; and also by Downes and Gautam, 1999, The G-Protein Subunit Gene Families. Genomics 62:544-552), the contents of both of which are incorporated herein by reference.
Known and uncharacterized GPCRs currently constitute major targets for drug action and development. There are ongoing efforts to identify new G protein coupled receptors which can be used to screen for new agonists and antagonists having potential prophylactic and therapeutic properties.
More than 300 GPCRs have been cloned to date, excluding the family of olfactory receptors. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Cudermann et al., J. Mol. Med., 73:51-63, 1995).
Formyl peptide receptor-like 2 (FPRL2) (SEQ ID NO: 1, human polynucleotide sequence, SEQ ID NO: 2, human amino acid sequence) is a member of FPR Family. The members of this family belong to the GPCR family. Human FPR (SEQ ID NO: 3, human polynucleotide sequence, SEQ ID NO: 4, human amino acid sequence) was first member of the FRP family defined biochemically, in 1976, as a high affinity binding site on the surface of neutrophils for the prototypic N-formyl peptide formyl-methionine-leucyl-phenylalanine (fMLF). It was then cloned in 1990, by Boulay et al. from a differentiated HL-60 myeloid leukemia-cell cDNA library [Boulay, F. et al. (1990) Biochem. Biophys. Res. Commun. 168, 1103-1109; Boulay, F. et al. (1990) Biochemistry 29, 11123-11133]. In transfected cell lines, FPR binds fMLF with high affinity (Kd<1 nM) and is activated by picomolar to low nanomolar concentrations of fMLF in chemotaxis and calcium ion (Ca2+) mobilization assays.
Two additional human genes, designated FPRL1 (FPR-like 1) (SEQ ID NO: 5, human polynucleotide sequence; SEQ ID NO: 6, human amino acid sequence) and FPRL2 (FPR-like 2), were subsequently isolated by low-stringency hybridization using FPR cDNA as a probe [Ye, R. D. et al. (1992) Biochem. Biophys. Res. Commun. 184, 582-589; Bao, L. et al. (1992) Genomics 13, 437-440] and shown to cluster with FPR on human chromosome 19q13.3 [Murphy, P. M. et al. (1992) J. Biol. Chem. 267, 7637-7643 ; Bao, L. et al. (1992) Genomics 13, 437-440]. FPRL1 is defined as a low-affinity fMLF receptor, based on its activation only by high concentrations of Fmlf (μM range) in vitro [Murphy, P. M. (1996) Chemoattractant Ligands and their Receptors (Horuk R, ed.), pp. 269-299, CRC Press, Inc., Boca Raton; Prossnitz, E. R. and Ye, R. D. (1997) Pharmacol. Ther. 74, 73-102]. However, it is unclear whether such concentrations of fMLF could be generated at sites of bacterial infection or tissue injury. Therefore, the role of FPRL1 as another bona fide functional fMLF receptor in vivo remains to be determined. FPRL2 does not bind or respond to N-formyl peptides [Durstin, M. et al. (1994) Biochem. Biophys. Res. Commun. 201, 174-179] but instead shares some non-formylated chemotactic peptides identified for FPRL1 [Christophe, T. et al. (2001) J. Biol. Chem. 276, 21585-21593 ; Betten, A. et al. (2001) J. Clin. Invest. 108, 1221-1228].
Although FPR and FPRL1 were initially detected in phagocytic leukocytes, other cell types also express these receptors but with undefined biological significance. Little information is available about the expression pattern of FPRL2, except that mRNA for this receptor is present in monocytes but not neutrophils [Durstin, M. et al. (1994) Biochem. Biophys. Res. Commun. 201, 174-179]. Functional FPRL2 is also expressed in mature dentritic cells (DCs) [Yang, D. et al. J. Leukoc. Biol. Vol. 72: 598-607 (2002)], which express reduced levels of FPR but do not appear to express FPRL1 [Yang, D. et al. (2001) J. Immunol. 166, 4092-4098 ; Braun, M. C. et al. (2001) Blood 97, 3531-3536].
The Heme Binding Protein (HBP) (Sequence ID No7: human polynucleotide sequence, Sequence ID No8: human amino acid sequence; Sequence ID No9: mouse polynucleotide sequence, Sequence ID No10: mouse amino acid sequence). The human and mouse HBP cDNAs are 567 and 570 bp long respectively and encode a protein product of 189 and 190 amino acids respectively. This protein is located into the cytoplasm of the cell. HPB binds heme and porphyrins with micromolar Kd. HBP may function as a buffer for overproduced porphyrin as well as heme. Expression studies indicated that the mouse mRNA encoding HBP is expressed in liver, spleen and kidney cells (Blackmon et al; 2002 Arch. of Bichem. and Biophysics 407, p196-201).